Monoclonal antibodies can influence tumor cell survival by several mechanisms, acting directly on the tumor target cells and/or by activation of effector mechanisms mediated by soluble factors or cells. Originally, antibody was of murine origin and, when administered to humans, induced strong immune responses to the foreign mouse immunoglobulin. This limited their use. Genetic engineering has enabled the development of so-called humanized antibodies with increased therapeutic efficacy.
Chimeric antibodies are 65-90% human nucleotides sequences and consist of the murine variable regions, which bring about antigen recognition, fused to the constant or effector part of a human antibody. Humanized antibodies are about 95% human, and are made by grafting only the hyper-variable region, or complementarity-determining regions, of the murine antibody—which determines antibody specificity—onto a human antibody backbone. The developments of genetically engineered transgenic mice and advances in the generation of synthetic human antibody libraries have enabled the production of fully human antibodies on a commercial scale.
Chimeric, humanized, and fully human antibodies have less immunogenicity than early antibody constructs and allow repeated antibody administration, an improved capacity to recruit cytotoxic cells and complement, and an increased stability in the circulatory system. These improvements have contributed to the increased therapeutic efficiency of monoclonal antibodies.
Small antibody fragments have also been engineered to improve antibody penetration into bulky avascular tumors, especially solid tumors. A single-chain fixed-variable (Fv) region consists of only one heavy-chain variable domain and one light-chain variable domain, which are covalently linked by a short peptide linker. These fragments can be used to deliver radioisotopes or drugs to tumor sites. Antibodies have also been designed that have two different antigen-binding arms and hence have dual-binding specificity. An example is when one arm binds to the tumor cell and the other binds to the effector cell. Another example is when the arms bind not only to two different antigens on the tumor cell, but also to the effector cell by their Fc fragment. These constructs increase antibody-mediated tumor-cell killing through the recruitment of host immune-effector cells (T cells, natural-killer cells, and macrophages).
Many antigens that are recognized by monoclonal antibodies are expressed not only by malignant cells, but also by at least one subset of healthy adult cells. The best target antigen for a monoclonal antibody (mAb) therapy would be one that is both stably and homogeneously expressed by all tumor cells, is not or hardly expressed by normal tissues, does not exist under a soluble form (to avoid rapid antibody clearance), and is easily accessible to the monoclonal antibody.
Unlabelled antibodies cause tumor cells to die or to stop their proliferation through a combination of different mechanisms: 1) recruitment and activation of effector cells by antibody-dependent cell-mediated cytotoxicity (ADCC), by complement-dependent cytotoxicity (CD C), 2) blocking of receptor-ligand interactions and prevention of growth factor activity, 3) induction of apoptosis, and 4) secretion of cytokines.
Some antibodies do not induce cell death by themselves and instead are used to deliver radioisotopes, toxins, enzymes, or drugs to tumor sites. Specific targeting of cytotoxic agents to tumor cells has the potential to reach high concentrations at tumor sites, without the dose-limiting side-effects of systemic administration. In that context, antibodies that target antigens that are rapidly internalized offer another advantage.
Radioimmunoconjugates selectively deliver radiation to tumor sites. In terms of radionuclides, one may use, for instance, I131 or Y90, as well as new β (212 Pb, mixte βγ 177 Lu, 153 Sm, 186 Re, 67 Cu, 225 Ac . . . ) or α (213 Bi, 211 At . . . ) emitters, advantageously using improved chelation technologies such as MX-DTPA, CHX-A″ DPA, C-DOTA, PA-DOTA, DOTA-NCS (2-p-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid). The cytotoxicity depends on the pharmacokinetics of antibody localization and retention of the radionuclide. Healthy tissues, especially bone marrow, are also irradiated because of strong emission energies.
Alpha chains of plant and bacterial toxins (such as ricin, diphtheria toxin, or pseudomonas toxin) can be directly attached to monoclonal antibodies. After binding and internalization in the cell, these toxins disrupt protein synthesis at low concentrations. These toxins also commonly elicit strong immune responses in humans, which limits their repeated use.
Drugs such as doxorubicin and calicheamicin can be directly attached to monoclonal antibodies. Antibody-mediated delivery with calicheamicin allows clinical use of this highly potent agent, which would otherwise be too toxic for systemic administration on its own.
For reviews on these aspects, see M. Harris (2004), and Cancer Highlights (2004).
CD71 is a type II glycoprotein which exists as a homodimer of 180 kDa, linked by a disulfide bond in position Cys89. This glycoprotein is acylated at Cysteine 62 and phosphorylated at Serine 24 by protein kinase C. It contains an internalization signal constituted by a tetrapeptide YTRF (amino acids 20-23) (Collawn et al, 1993). Upon cleavage between Arg 100 and Leu 101 by a yet unknown protease, CD71 becomes soluble. O-glycosylation at Thr 104 reduces the sensitivity of CD71 to cleavage. Mammalian and chicken transferrin receptors have an RGD sequence, suggesting a possible evolutionary relationship with adhesion molecules. In terms of sequence homology, there is an homology with the C5a streptococcal peptidase sequence and with the PSA sequence (Prostate Specific Antigen, which is not prostate-specific but which exhibits a type II acid dipeptidase activity). A ligand of CD71 is the transferrin, protein responsible for iron transport. Recently, it has been shown that CD71 is also a receptor for IgA (Haddad et al, 2003).
Ferrotransferrin binds to CD71 under neutral pH and is internalized in the endosomal compartment where the pH is about 5. Iron is released and carried in the cytoplasm by an unknown mechanism. The apotransferrin remains bound to CD71 at pH5 and returns to the cell surface where the pH is about 7.4. Under neutral pH conditions, the apotransferrin has no longer affinity for CD71, allowing thus another cycle to begin. This feature of CD71 permits to use this surface molecule for internalizing a drug, a toxin or a radioelement coupled to an antibody (Lee et al, 2000) (Li et al, 2002) (Ng et al, 2002) (Shinohara et al, 2000). CD71 binds in cis, via a non-covalent binding, to the ζ chain of the TCR, where it may be involved in signal transduction. Moreover, CD71 monomers are supposed to form a complex with integrin CD29/CD49d (VLA4) via a disulfide bond.
CD71 plays an essential role in cell proliferation by controlling iron uptake which is essential in several metabolic pathways. This occurs via the binding and the endocytosis of transferrin. Expression of CD71 is post-transcriptionally regulated through RNA stability; it also depends on iron intracellular levels. The IRE-BP (iron-response element binding protein) exists under two different form, IRP-1 and IRP-2. IRP-1 is similar to the mitochondrial aconitase, whereas no aconitase activity has yet been described for IRP-2, which is however structurally homologous to aconitase. In case of iron deficiency, IRP-1 and IRP-2 stabilize the CD71 RNA by binding to specific sequences designated IREs (iron response elements located in the untranslated 3′ region of the RNA). When the level of iron is high, the IRE-BP affinity for IREs is low and the RNA is more sensitive to degradation. In addition, nitric oxide modifies CD71 expression by activating the binding of IRE-BP and by stabilizing the RNA.
CD71 is almost undetectable on leucocytes and resting cells. This expression is up-regulated by cell activation and proliferation. For these reasons, CD71 appears to be a target of choice for treating a number of tumors. Nevertheless, in the red blood cell line, red blood cell precursors and reticulocytes express CD71. This is also deduced from mice lacking the CD71 transferrin receptor, which have a severe phenotype affecting both erythropoiesis and neurologic development (Levy et al., 1999). This was also shown in human in a case of an acquired iron-deficiency anemia by Larrick J. W. and Hyman E. S., 1984. On the contrary, this showed that stem cells do not express CD71, confirming that an anti-CD71 antibody may be useful for anti-tumor therapy (Zech et al, 2003). Besides, CD71 is expressed on the cerebral endothelium and this enables drugs to go through the blood-brain barrier, especially for treating gliomas (Lee et al, 2001). Finally, this last property, if deleterious under given circumstances, can be inhibited upon simultaneous administration of chloroquine.
Metastatic melanoma is a tumor with very poor prognosis, whose incidence is increasing due to modern life style and to prolonged, repeated and unprotected exposure to the sun. At least about 6000 new cases are reported per year in France, with an incidence of 9/100,000 inhabitants in Paris and its region. One third of these cases occur when the subject is less than 40 years old. The prognosis is closely related to the depth of the initial tumor and it results in death in about 100% of the cases of metastatic melanoma. Since more than 20 years, various therapies, either conventional or more innovative, have been tested, but they neither modified the prognosis nor changed the fatal outcome of metastatic melanoma. Despite a better general knowledge, including the identification of tumor antigens and auto-reactive CTL, immunotherapy remains unsuccessful. Immunotherapy has associated in various ways tumor cell vaccination with tetramer-associated peptides, adjuvants, cytokines or dendritic cells, but has remain inoperative. This may be explained at least in part by a host immunodeficiency (Ugurel S, 2004). Monoclonal antibodies targeting antigens restricted to melanomas such as antigangliosides GD2, GD3, GM2, p97 melanotransferrin, p240 antigen of high molecular weight, have been used (Noronha E J, 1998). Clinical studies have shown that these antibodies can be administered with minimal toxicity. Nevertheless, to date, no antibody, even humanized, chimeric or human (Mills L, 2002), used alone or in combination with a toxin (Shinohara H, 2000), a chemotherapeutic agent, a growth factor such as GM-CFS, IL2 interleukin (Soiffer R J, 1997), TNF or interferon, has proven to be really successful.
More specifically, choroidal and cutaneous melanoma both came from neural crest. Very few people are affected by choroidal metastasis melanoma. These melanomas differ from each other in two points: dissemination mode and the affected organs by metastasis. Thus, choroidal metastasis melanoma dissemination is exclusively by haematogenous way. In more than 90% cases, the first metastasis site (often unique) is hepatic. Few patients will develop other tumor localizations and a majority of them will die in the nest 6 months after wide hepatic metastasis diagnosis. Cutaneous melanoma presents an evolutionary mode extremely variable and unpredictable because of dissemination is both haematogenous and lymphatic. There is a more specific tropism for the brain although the other organs can be touched. Diagnostic of these two melanomas is mostly made at a localized stage. The associated treatment is then local: it consists of a surgical act or a radiotherapy for the choroidal melanoma and surgical for the cutaneous melanoma.
As mentioned above, it is important to stress that except the surgery and the radiotherapy, conventional therapeutics applied as part of adjuvant treatments or for metastasis disease are very limited in term of efficiency. Moreover, these treatments, requiring prolonged hospitalizations, are painful, heavy and mostly expensive. It thus seems necessary to develop, beside conventional treatments, innovative approaches such as the immunotherapy. The therapeutics, beside local treatments (surgery, chemo- and radiotherapy), are inexistent for choroidal metastasis melanoma and very limited for the cutaneous metastatic melanoma. Therefore, new approaches must be developed to increase the survival of patients affected by metastatic melanoma.